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Introduction

Y-STR testing is an established tool in the forensic casework, paternity testing and genealogy communities. Commercially available kits for Y-STR testing include the PowerPlex® Y System with 12 Y-STR loci and Applied Biosystems AmpFlSTR® Yfiler® kit, which includes the PowerPlex® Y loci plus five additional loci for a total of 17 Y-STR loci. There has been a significant amount of research
(1)(2)(3)(4)(5)(6)
into additional Y-STR loci that may increase the ability to discriminate between individuals. The PowerPlex® Y23 System includes six of these Y-STR loci. In addition, the PowerPlex® Y23 System, while optimized for extracted DNA and casework amplifications, allows direct amplification from FTA® card punches, pretreated swabs and pretreated punches from S&S 903 paper or Bode Buccal DNA Collector™ devices.

The PowerPlex® Y23 System

The PowerPlex® Y23 System (Cat.# DC2320 and DC2305) is a five-dye multiplex system that combines the 17 Y-STR loci currently included in Y-STR kits (DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y-GATA-H4) with six new highly discriminating Y-STR loci (DYS481, DYS533, DYS549, DYS570, DYS576 and DYS643). See Figure 1. The system includes the same dyes as the other Promega five-dye systems (e.g., PowerPlex® ESX, ESI, 18D and 21 Systems), and amplification products can be detected using the Applied Biosystems® 3100, 3130 or 3500 Genetic Analyzer. The PowerPlex® Y23 thermal cycling program requires one hour and 40 minutes for extracted DNA (90 minutes for direct amplification), allowing rapid turnaround of results.

Gene Diversity and Mutation Rates

Vermeulen et.al(2)
analyzed 67 Y-STR loci with a global set of male DNA samples. From that study, the six additional loci included in the PowerPlex® Y23 system were considered the most informative for differentiation of male haplotypes. One potential reason for the added discrimination of these loci is their mutation rates. With the exception of DYS643, these additional Y-STR loci exhibit relatively high mutation rates, with the mutation rates for DYS570 and DYS576 estimated to be >1% . The gene diversities of loci included in the PowerPlex® Y23 System are shown in Table 1. The high gene diversities of the six additional Y-STR loci included in PowerPlex® Y23 increase the ability of this system to distinguish individuals. The high mutation rates contribute to more allelic diversity with these loci and thus a better ability to distinguish between family members. Of course, these higher mutation rates must be taken into consideration for paternity testing or missing persons cases in which other family members may be used as references.

Table 1. Gene Diversity of Loci Included in Commercially Available Y-STR Systems.

Locus

Gene Diversity

PowerPlex® Y System

AmpFlSTR® Yfiler® Kit

PowerPlex® Y23 System

DYS643

0.92(5)

√

DYS570

0.85(5)

√

DYS481

0.85(2)

√

DYS385 a/b1

0.83(6)

√

√

√

DYS576

0.82(5)

√

DYS549

0.78(5)

√

DYS458

0.78(6)

√

√

DYS390

0.75(6)

√

√

√

DYS533

0.75(5)

√

DYS635

0.71(6)

√

√

DYS456

0.70(6)

√

√

DYS448

0.69(6)

√

√

DYS438

0.68(6)

√

√

√

DYS439

0.67(6)

√

√

√

DYS392

0.64(6)

√

√

√

DYS19

0.64(6)

√

√

√

DYS437

0.62(6)

√

√

√

DYS389II

0.62(6)

√

√

√

Y-GATA-H4

0.60(6)

√

√

DYS389I

0.56(6)

√

√

√

DYS391

0.53(6)

√

√

√

DYS393

0.46(6)

√

√

√

Amplification of Extracted DNA and Casework Samples

The PowerPlex® Y23 System is optimized for amplification of 0.5ng of extracted DNA (Figure 2). However, the PowerPlex® Y23 System is a robust system that allows amplification of small amounts of male genomic DNA in the presence of excess female genomic DNA. Figure 3, Panel A, shows amplification of 125pg of male genomic DNA, and Panel B shows amplification of 125pg of male genomic DNA with 2,000ng of female genomic DNA. This figure shows a full Y-STR profile in a sample with a 16,000-fold excess of female genomic DNA. Peak heights showed only a slight decrease with the excess female genomic DNA.

Figure 2. Amplification of 0.5ng of human male genomic DNA. DNA was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

DNA was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

Amplification of Database Samples

The PowerPlex® Y23 system is compatible with direct amplification of DNA from blood or buccal samples on FTA® card punches (Figure 4). The PowerPlex® Y23 System can be used with S&S 903 paper and Bode Buccal DNA Collector™ punches that have been pretreated with the Promega PunchSolution™ Kit (Cat.# DC9271) as well as swabs that have been pretreated with the SwabSolution™ Kit (Cat.# DC8271). See Figure 5.

Figure 4. Direct amplification of DNA on an FTA® card punch. DNA from one 1.2mm FTA® card punch containing blood was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

DNA from one 1.2mm FTA® card punch containing blood was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

Figure 5. Direct amplification of DNA from a swab. DNA from a buccal swab pretreated with SwabSolution™ Reagent was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

DNA from a buccal swab pretreated with SwabSolution™ Reagent was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

Conclusions

PowerPlex® Y23 is a robust system that can be used for amplification of extracted DNA as well as direct amplification. The system amplifies six new highly discriminating Y-STR loci, making it more discriminating than currently used Y-STR systems. The system also amplifies low amounts of male genomic DNA in the presence of a large excess of female genomic DNA. The rapid thermal cycling conditions significantly reduce the time required to obtain a Y-STR profile, streamlining laboratory workflows.

Contribution of an article to Profiles in DNA does not constitute an endorsement of Promega products.

PowerPlex is a registered trademark of Promega Corporation. PunchSolution and SwabSolution are trademarks of Promega Corporation.

AmpFlSTR and Yfiler are registered trademarks of Applera Corporation. Applied Biosystems is a registered trademark of Applied Biosystems. FTA is a registered trademark of Flinders Technologies, Pty, Ltd., and is licensed to Whatman.

Products may be covered by pending or issued patents or may have certain limitations. More information.

All prices and specifications are subject to change without prior notice.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.

Figures

Figure 2. Amplification of 0.5ng of human male genomic DNA. DNA was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

DNA was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

Figure 4. Direct amplification of DNA on an FTA® card punch. DNA from one 1.2mm FTA® card punch containing blood was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

DNA from one 1.2mm FTA® card punch containing blood was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

Figure 5. Direct amplification of DNA from a swab. DNA from a buccal swab pretreated with SwabSolution™ Reagent was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

DNA from a buccal swab pretreated with SwabSolution™ Reagent was amplified and amplification products were separated on an Applied Biosystems® 3130 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex® Y23 System Technical Manual TMD035.

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